...Fab fragment, it either needs to be directly conjugated to a reporter or you need a light-chain specific...antibody. Fab fragments are useful when you just need an antibody’s antigen binding function but not the...antibodies, you can make your own antibody fragments as needed.  In contrast, anti-IgG antibodies, commonly used...fragments make good secondaries for cases where you need to pre-form primary-secondary antibody complexes...about what pieces of the antibody you do and don’t need, and how the protein will interact with other components...

...stabilized promoters incorporate feedback loops such as the incoherent feedforward loop (iFFL). In general, iFFLs...to interpret or can interfere with the precision needed in metabolic engineering or other synthetic biology...deoxychromoviridans requires three genes whose proteins are needed sequentially. When Segall-Shapiro et al. placed... In cases where a specific ratio of products is needed, such as within a multi-protein complex, stabilized...between the different components. When your pathway needs precise fine-tuning of gene expression levels, give...

...primers, or mammalian selection markers. You don’t need a fancy database to organize your own plasmid collection...of your lab can access and use the materials they need, and avoid duplicating your effort. In addition,... it will be easy to find all of the information needed for the process. Always sequence verify Once the...sometimes more depending on the number of features we need to verify. Determining which features to verify ...Remember, when designing a sequencing primer, you need some space between your primer binding site and ...high-throughput plasmid sequencing If you’re like us, you need a strategy for processing several sequencing reactions...

...deposit process. To deposit plasmids online, you will need to create an account with Addgene before starting...depositing 10 or more plasmids. (If you believe you need to deposit a collection that is greater than 75 ...contains columns that correspond to the plasmid data we need; you can copy and paste your information directly...antibiotics, etc.) and stored as glycerol stocks We will need information on how to verify any genomic alterations...modify the gene/insert and alternative name boxes as needed. Use the "Relevant mutations/deletions" box to ...entry fields to describe the next insert. Repeat as needed for more inserts. When you are finished entering...addgene.org. Include your deposit number and which data needs to be updated. When your plasmids arrive at Addgene...

...many cases where the specific viral particles you need are not available, or the cost of custom viruses...too high for your budget. In those cases, you’ll need to produce your viruses in-house.   Introducing...trying to make. For example, bacteriophages will need to be produced in bacteria cells. However, some ... producing a stock P1 virus, so that if you ever need to remake the same virus, you can skip the first... just two days, but add additional costs and may need to be optimized for your purpose. Making your decision... Baculovirus; can produce AAV and lentivirus if needed # of plasmids Three or four Two Packaging limit...

...start-ups are turning to software apps to solve our need for quicker, more effective updating and dissemination...protocols when they begin their own experiments. Need to submit an updated protocol? Just email us at ...departments, industry, and more to fit every lab's needs. Plus there are the apps - many to help you access...solution to your lab's organization and management needs. With Quartzy you can share protcols with your labmates...your thoughts on how the app works for your lab’s needs. At Addgene, we’ll continue to create new molecular...and wikis - by Rory Macneil on Bitesize Bio Data Freedom: The Expansion of Data Sharing in Research Publications...

...customs processes, and the distances the packages needed to travel meant the accessibility varied country...showing exactly how to execute a step can provide needed clarity (Table 1). Our most popular video, however...measure of our educational resources’ value is feedback from the researchers who use them. The best part...out to ask if they can help us create or update a needed resource, and when they tell us how they use our...to enable virtual learning during the pandemic. Feedback like that lets us know that Addgene’s materials...internationally, after all, that we could identify the need for distributors in some countries. ‌We’ve developed...

...own expression, so high levels of DnaA act as a feedback mechanism to reduce further transcription. Replication.... Fun fact! As far as we can tell, DnaA doesn’t need to hydrolyze ATP to do its job — although ATP does...for a given plasmid has been reached, negative feedback circuits reduce replication of the plasmid to ...plasmid ori, after which replication of the plasmid proceeds (and terminates) similarly to the bacterial chromosome...controls the copy number of pSC101 through a negative feedback cycle. As with DnaA, high concentrations of RepA...given plasmid influences how high the copy number needs to get before handcuffing occurs. These mechanisms...

...this problem in C. roseus by working with young seedlings and optimizing several other aspects of the transient...D'Aoust et al., 2009): Usually a syringe without a needle is used to inject an Agrobacterium culture through... a great help in screening candidate genes and speeding up research. Certainly, there is still more to...Ching-yeh, et al. "Intrinsic GUS-like activities in seed plants." Plant cell reports 9.1 (1990): 1-5. PubMed...method for transient gene expression analysis in seedlings of Arabidopsis and other plant species." Plant...analyzing gene function in Catharanthus roseus seedlings." Frontiers in plant science 10 (2019). PubMed...

... and by offering advice on areas where you might need some help. Today: Career Planning Resources. The...be a graduate student or postdoc forever, and you need to be thinking about what comes next. To that end...attending one of these fairs is a great start, but you need to do more than just show up. Do your homework and...employer in mind, you may consider starting with indeed.com or monster.com, where you’ll find a variety of...hope to have a career outside of academia, you may need to look a bit harder to find your mentor. This may...even be a colleague. A mentor does not necessarily need to be someone a lot older than you are; try using...

... of each plasmid needed for the whole experiment Total volume (μL) of plasmid needed for the whole experiment...specific molar ratios. To get these ratios right, you need to do a few calculations. This calculator can do...will calculate the following μg of each plasmid needed per dish or stack. Calculations are based on using...equation the calculator uses to give you the μl needed of each plasmid. Each part of the equation is labeled...

...ll cover the normalization and loading controls needed for relative quantification of a western blot —... it’s overkill in many cases. Often, you may not need to know the exact quantity of your POI, but simply...normalization is a nearly ubiquitous tool, but proceed with caution! Loading controls only address one...your context. If you don’t find anything, you’ll need to conduct a control experiment to demonstrate that... in a separate step from the POI blot, you don’t need to worry about the linear detection ranges of multiple...increasing concentration of β-actin in panel A. Indeed, the quantification in panel B is not especially...

... in almost all organisms and cell types, (ii) no need to add a co-factor, (iii) easy visualization with...EGFP variant, mEGFP (Zacharias et al., 2002). GFP needs oxygen Chromophore formation requires a fully folded...specific biological system under anaerobic conditions needs to be examined on a case-by-case basis. GFP is sensitive...light by a chemical reaction and hence, there is no need to excite the sample. Although bioluminescent probes...for biological spectroscopy and microscopy." Proceedings of the National Academy of Sciences 114.23 (2017... real-time multicolor luminescence imaging." Proceedings of the National Academy of Sciences (2015): 201418468...

...narrative. Scientists do this all the time, you just need to figure out how to make it suspenseful. 4. Start...your talk early - like today Don’t wait until you need to speak to start creating slides or thinking about...will be familiar with the terms. If you feel you need to define a term, but are worried that this might...but not reading the explanation. The people that need the information will find it. Experienced speakers...mentoring groups to practice giving talks and getting feedback. Wouldn’t you rather hear it from your peers than... many things. One good topic or story is all you need, if you do it well.  Resist the urge to show all...

... scientists by bringing you all the plasmids you need. We're focused on our mission – documenting your... for scientists everywhere to get the tools they need to progress their research. *2015 total reflects... our growing community! Our plasmid repository succeeds because of our amazing community. The trust of...transfer offices that facilitate MTAs. The great feedback from scientists who request plasmids and use our...from our blog readers. You all drive Addgene to succeed and to improve, so thank you. 500,000x Thank You...

...do you decide which tools to use, and what do you need to know about them to be successful in your experiments...to activate or inhibit specific neurons without needing to consider secondary signaling pathways. There... of tool appropriate for your experiment, you’ll need to find a specific tool. There are many, many options...times or frequencies (Zhang & Cui, 2015). You’ll need to test for these differences when designing your...previously published system in your own work, you’ll need to know (and understand!) the hardware types, construction...light-gated cation-selective membrane channel. Proceedings of the National Academy of Sciences, 100(24),...

...have been accumulated over the years using them. Indeed, the future of mouse work is bright as it is now... a mouse model, and its success is not always guaranteed. To learn more about this process check out our...Part 1: Genetically Engineered Mice and Part 2: Breeding and Crossing Mice. My first CRISPR mouse experiment...oligonucleotide or plasmid as the repair material if needed (for knock in models). CRISPR mouse model basics...directly into fertilized mouse eggs, circumventing the need for enrichment and selection offered by mouse ESCs...transmission of the transgene, this technique is still in need of improvement. Also when working with CRISPR, one...

...generation of tools had limited sensitivity and speed and were only available as green probes. The Patriarchi...important because alpha2-AR can provide inhibitory feedback in response to norepinephrine release, and clinically-used...however, be studied with the nLight indicators. Speed One of the major advantages of this next generation...generation of norepinephrine sensors is their speed. nLightG is activated eight times faster than GRABNE and...concentrations of dopamine. This cross reactivity will need to be accounted for in your experimental design....

... are not convinced by the existing data, you may need to roll up your sleeves and perform some extra validation...that it will perform as expected in another. You’ll need to validate that points 1 and 2 hold true under ...different tissues or cell types. If your antibody indeed binds to your target then you would expect to see...expression and non-specific, background signal, so you’ll need a range of expression strengths to be confident ...unexpected. This approach has high specificity, but you need to be aware of any possible alternative translation...identified as your target protein, allowing you to proceed with confidence.  Share your results Ultimately...

...information about. The assumptions and requirements that need to be met for a parametric test are as follows:   ...test as the standard value has no variation that needs to be accounted for. ANOVA tests ANOVA tests are...these tests. All sample comparisons Do you really need to know every pairwise comparison within your population...samples are assessed. However, if many comparisons need to be performed, Bonferroni and Benjamini will greatly... appropriate to use if all sample comparison is needed, without a high number of samples (~5). Tukey’...instead of after you’ve already run it.) You don’t need to be a mathematician to select the right statistical...